Carboxylate-gated-nitroxide (CGN) compounds and compositions and methods of use thereof

ABSTRACT

Carboxylic-gated-nitroxide (CGN) compounds and their esterified derivatives are discovered and disclosed as compositions and shown to have the potential in treating a variety of acute and chronic diseases and disorders resulting from reactive oxygen species (ROS) injury. Compositions for treating tissue damage from ROS injury containing CGN, or active derivatives thereof, in a suitable pharmaceutical, cosmetic, and diagnostic formulations are described.

CROSS-REFERENCE

This application is a continuation-in-part of our earlier filed U.S.patent application Ser. No. 09/948,505, filed Sep. 6, 2001, nowabandoned, which application is incorporated herein by reference and towhich application is claimed priority under 35 U.S.C. §120.

FIELD OF THE INVENTION

This invention relates to the treatment and prevention of chronic andacute diseases and disorders originated from oxidative stress. Novelcompounds, compositions and their methods of use also relate to thefield of therapeutic and diagnostic pharmaceuticals.

BACKGROUND OF THE INVENTION

Many acute and chronic diseases and disorders are attributable to theinjuries from endogenously and exogenously generated reactive oxygenspecies (“ROS”). ROS injuries occur when the normal detoxifyingcapability of antioxidant enzymes and vitamins are overwhelmed.Supplements containing recombinant antioxidant enzymes and vitaminstreating ROS injuries have revealed some advantages and limitations.Nitric oxide (NO•) and superoxide (O₂•⁻) are both physiological gaseousfree radicals which alone or in combination are capable of initiatingthe ROS cascade and injuries. Onging drug development programs targeteither supplementing or removing NO•. Using superoxide dismutase (SOD)as a model, an alternative drug development strategy is to target theremoval of O₂•⁻ and attenuation of follow-on ROS cascade. The latterprograms involve mimicking the catalytic activities of SOD in a“metal-center” or a “metal-free” synthetic molecule such as chelatedtransition metal ion or nitroxide, respectively. The use ofpolynitroxylated “metal-center” (i.e. polynitroxyl hemoglobin) and“metal-free” (i.e. polynitroxyl albumin) blood proteins as vascular ROSinjury protectants and imaging agents is described in patents such asU.S. Pat. Nos. 5,725,839; 5,741,893; 5,767,089; 5,804,561; 5,807,831;5,817,632; 5,824,781; 5,840,701; 5,591,710; 5,789,376; 5,811,005;6,048,967. However, it is generally accepted that no one approach cantreat all ROS injuries. In fact, continued research efforts have beenrequired to address new challenges not previously envisioned. Thepresent invention shows the existence of a new synthetic analog of a“carboxylate-gated-nitroxide (CGN)” which is a center of activity.

The present invention also shows synthesis of CGN and its “metal-center”and “metal-free” novel compounds in esterified and non-esterified formsand their utility in targeted delivery to therapeutic sites of interest.In addition, conjugation via covalent linking and conjunctive use withexisting drugs or targeting molecules increase their therapeuticapplications.

SUMMARY OF THE INVENTION

Carboxylate-gated-nitroxide (CGN) compounds, their esterifiedderivatives compositions and methods of use for the treatment andprevention of diseases and disorders from ROS injuries are disclosed.Compositions of the invention are comprised of CGN and are useful fortreating tissue exposed to undesirably high levels of reactive oxygenspecies (ROS). In addition to CGN, it is possible to use activederivatives thereof, in a suitable pharmaceutical, cosmetic, anddiagnostic formulation for intravenous, i.p, i.m., oral, topical, nasal,pulmonary, or rectal administration. Examples of CGN and its derivativesinclude Esterified CGN, Esterified Dicarboxylic Amino acid CGN, andEsterified Carboxylic di-Amino acid CGN and their derivatives, includingdi-nitroxide, tri-nitroxide derivatives, or conjugates used alone or incombination with existing drugs or targeting molecules. Pharmaceuticalformulations and routes of administration of the nitroxides aredescribed. In particular, topical applications of compositions of theinvention show how the targeted delivery of the CGN intracellularly isachieved through cellular esterase cleavage of hydropobic esterified CGNas a pro-drug to a hydrophilic CGN in its free acid form.

Nitroxide compounds of the invention comprise three molecular featureswhich provide inventive characteristics. First, compounds of theinvention comprise a first portion of a molecule comprising a nitrogenand an oxygen atom bound directly together and comprise an unpairedelectron (•NO). Second, compounds of the invention comprise a secondportion of the molecule which comprises at least two oxygen atoms, whichsecond portion provides a negative charge, e.g. one, two, three or morecarboxylic acid groups. The second portion may be structured so as tocreate the carboxylic acid groups after a given reaction e.g. the alkylmoieties of ester groups are removed. Third, compounds of the inventioncomprise a linking group positioned between the first and secondportions which linking group allows the negative charge effect of thesecond portion to “bend toward” the first position and thereby aid inmaintaining the stability of the (•NO) portion in a range ofenvironments in a human body.

The compositions and routes of administration of the CGN are describedin topical applications to show how the targeted delivery of the CGNintracellularly is achieved through the cellular esterase cleavage ofhydropobic esterified CGN as a pro-drug to a hydrophilic CGN in its freeacid form. Improvement of biological half-life and intracellulardelivery over 4-hydroxyl-2,2,6,6-tetramethyl-piperidine-1-oxyl, a priorart compound, is definitively demonstrated using nitrogen stableisotopes of the two nitroxides and their co-administration (aerosol,rectal, and oral) distribution, metabolism and elimination studies asdetected by electron paramagnetic resonance spectroscopy. This inventionshows that esterase hydrolyzed esterified CGN provide their carboxylicacid moiety as the “gate” which protects the in vivo stability of themolecule without affecting catalytic activity. Examples of topical,i.v., oral, and pulmonary administration presented include the selectedderivatives of CGN. The examples of efficacies of CGN in the preventionof acute and chronic skin lesions induced by UVB when applied pre- andpost radiation are presented.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the generic structural formula of esterifiedcarboxylate-gated nitroxide (CGN) and their derivatives.

FIG. 2 shows the generic structure formulation of esterifieddi-carboxylic amino acid CGN

FIG. 3 shows the generic structure formulation of esterified carboxylicdi-amino acid and their derivatives

FIG. 4 shows the generic structures of the selected compounds, which areexamplified in example section

FIG. 5 shows the generic compound2,2,6,6-tetramethyl-1-oxyl-piperidinene-4-succinate.

FIG. 6 shows the semi-generic structures of FIG. 5 with R₂ replaced byone hydroxyl (—H) group.

FIG. 7 shows the structure of FIG. 5 with R₂ and R₃ replaced by hydrogenreferred as the compound2,2,6,6-tetramethyl-1-oxyl-piperidinene-4-succinate (TOPS).

FIG. 8 shows the structure of FIG. 5 with R2 and R3 replaced by H andethyl respectively referred as the compound ofmonoethyl-2,2,6,6-tetramethyl-1-oxy-4-piperidylidene-succinate (E-TOPS).

FIG. 9 shows the structure of FIG. 5 where the both R₂ and R₃ groups arean ethyl group, i.e. theDi-ethyl-2,2,6,6-tetramethyl-1-oxy-4-piperidylidene-succinate (DE-TOPS)

FIG. 10 shows the structure of FIG. 5 where the both R2 and R3 groupsare tert butanol, i.e. the Di-tert-But2,2,6,6-tetramethyl-1-oxy-4-piperidylidene succinate (DB-TOPS).

FIG. 11 shows the semi generic structure of FIG. 5 where R₂ is Tempol,i.e. TE-TOPS.

FIG. 12 shows the structure of FIG. 5 where R₂ and R₃ are both Tempoli.e. TT-TOPS.

FIG. 13 shows the structure of FIG. 4 where the generic circle is a fivemembered ring.

FIG. 14 shows a liquid chromatograph spectrum identifying TOPS and itsester(s) of carboxylic acid derivatives, E-TOPS and DE-TOPS.

FIG. 15 shows the hydrolysis of DE-TOPS to TOPS by sodium hydroxide(NaOH) in an aqueous environment. As is apparent from the EPR signals,pre- and post-hydrolysis, near complete conversion from DE-TOPS to TOPSoccurs.

FIG. 16 shows liquid chromatograph HPLC and EPR spectra describing thestructure changes during the synthesis of TE-TOPS from ¹⁴N E-TOPS and¹⁵N Tempol.

FIGS. 17(A and B) shows the hydrolysis of ¹⁴N-¹⁵NTE-TOPS to two monoradical in vitro and in vivo.

FIG. 18 shows the extended in vivo plasma half-life of ¹⁴N E-TOPScompared to ¹⁵N Tempol when both are co-administrated intraperitoneallyin mice.

FIG. 19 shows the extended in vivo plasma half-life of ¹⁴N E-TOPScompared to ¹⁵N-Tempol when both are co-administrated intravenously inmice.

FIG. 20 shows the extended in vivo plasma half-life of ¹⁴N-E-TOPScompared to ¹⁵N-TEMPOL when both are co-administrated intramuscularly inmice.

FIG. 21 shows the extended in vivo plasma half-life of ¹⁴N-E-TOPScompared to ¹⁵N-Tempol L when both are co-administrated orally in mice.

FIGS. 22A–C shows skin penetration and compartmentalization of DE-TOPSin a Franz Diffusion cell when ¹⁴N-DE-TOPS and ¹⁵N-Tempol are co-appliedon hairless mouse skin: 22A—before application; 22B—in receptor cell;and 22C in skin at 20 hours.

FIG. 23 shows human skin penetration and compartmentalization of DE-TOPSby EPRI.

FIG. 24 shows the relationship between the survival rate and dosage forE-TOPS compared to Tempol in an LD₅₀ study in mice.

FIG. 25 shows the metabolism of DE-TOPS through excretion in urine andreduction in plasma after 10 day daily topical application.

FIG. 26 shows the superoxide dismultase activity of E-TOPS determined byEPR.

FIG. 27 shows that DE-TOPS, E-TOPS, and TOPS dose dependent inhibitionof hemoglobin-induced toxicity of cortical neurons.

FIG. 28 shows TOPS, E-TOPS, and DE-TOPS dose dependent inhibition ofperoxynitrite-induced toxicity on cortical neurons.

FIG. 29 shows that the nitration of hydroxy phenol acetic acid (HPA) byperoxynitrite was inhibited by TOPS, E-TOPS, and DE-TOPS in adose-dependent manner.

FIG. 30 shows that E-TOPS reduces apoptosis induced by tumor necrosisfactor alpha (TNF-α) on Y-79 cell line at different incubation times.

FIG. 31 shows that pre-topical application of DE-TOPS prevents UVBinduced skin damage on hairless mice acutely.

FIG. 32 shows the histopathological change of the hairless mouse skinafter 11 days of UVB radiation with control or DE-TOPS applyingpre-radiation

FIG. 33 shows that pre-topical application of DE-TOPS prevents UVBinduced skin damage on hairless mice chronically at 4 month.

FIG. 34 shows that post-topical application of DE-TOPS prevents UVBinduced skin damage on hairless mice acutely

FIG. 35 shows the scores of the histopathological change of the hairlessmouse skin after 11 days of UVB radiation with control or DE-TOPSapplying post-radiation

FIGS. 36(A, B and C) the comparison of effect of DE-TOPS with that ofTempol on UVB induced acute damage when applying 10 min or 2 hour preradiation.

FIG. 37 (three panels) shows a comparison of DE-TOPS, a placebo, andRetin A 15 days after treatment with DE-TOPS and ultraviolet radiation.

DETAILED DESCRIPTION OF THE INVENTION

Before the present compounds, formulations and uses are described, it isto be understood that this invention is not limited to particularstructures, formulae and uses described, as such may, of course, vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anitroxide” includes a plurality of such compounds or functional groupscells and reference to “the formulation” includes reference to one ormore formulations and equivalents thereof known to those skilled in theart, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Further, compounds of the invention may be structured so that it reactswith a biological organism it is provided to in a manner with changesthe structure of the compound thereby enhancing cite directed deliveryof the compound to a desired location.

Compounds of the invention are specifically designed for increaseddiagnostic and therapeutic utility when applied in a biological contextwhere intracellular retention is important and where hydrophobicbarriers such as skin, stratum corneum, or other such membranes areselectively permeable. These compositions are particularly useful whendelivered to esterase-containing cells. These compositions may be usedto alleviate the toxic effects of free radicals in a living organismthat result from exposure to chemical agents and toxins, as well as sun,UV, or other forms of ionizing radiation. Specialized methods andformulations also enable the compounds of the invention to diagnose andtreat a wide variety of physiological conditions, and to analyze in vivoreactions with imaging techniques that measure nitroxides and theirreactivity. The invention also relates to novel nitroxide compositionsthat permit targeting or compartmentalization of a therapeutic dose ofnitroxide within a localized area, particularly penetration andretention within an intra-dermal interface in the skin.

Compounds of the invention may be defined by the structural formulae asshown in FIGS. 1–13. These compounds can be formulated in any desiredmanner e.g. to create any desired pharmaceutically effectiveformulation. The compound may be mixed with, dissolved and/or suspendedin a pharmaceutically acceptable carrier. The carrier may be chosenbased on the intended use. For example, creams, gels and lotions may beused for topical applications to the skin. Saline solutions may be usedas injectables.

The compounds and compositions of the invention may be applied in anydesired manner. In general, compounds are formulated into compositionssuitable for a particular type of administration e.g. lotions applied tohuman skin, aqueous/ethanol formulation to gargle in the mouth and upperthroat, an aqueous saline solution for injection.

Invention in General

There are many aspects to the invention including compounds,formulations comprised of the compounds in a suitable carrier, andmethods of treatment. The compounds are structured to provide theantioxidation effect of CGN to a biological organism (e.g. human) and toprovide that effect over a sufficiently long period so as to obtain adesired therapeutic result. CGNs of the invention are structured in amanner such that the NO group is “gated” by carboxylate within the samemolecule so that its redox modulation function is maintained over alonger period of time in vivo. The NO group of CGN may be symmetricallyor asymmetrically positioned relative to 2 carboxylate groups.

Compounds of the invention (see FIGS. 1–13) can be combined into a widerange of formulations for a range of different treatments. The treatmentis generally a modulation of a biochemical reaction or more specificallya lessening of oxidation reactions as described further here. Themodulation effects obtained are generally greater than the antioxidanteffects of vitamins such as vitamin C and generally less than theeffects of endogenous catalysts. The need for modulating oxidationreactions is often created by the human body's overreaction toinfections as described generally below.

The blood is made up of plasma, red blood cells and white blood cells.The red blood cells allow for oxygen transportation and the white bloodcell provide a defense against infection. White blood cells includeleukocytes which include two types—lymphocytes and monocytes. Whiteblood cells also include granulocytes which are divided into threetypes—basophils, eosinphils and neutrophils.

The neutrophils are the most abundant of the white blood cells and ofparticular interest as regards the present invention. The neutrophilssqueeze through the capillary walls to find infected tissue or what maybe mistaken as infected tissue which has been subjected to trauma.Neutrophils are released from bone marrow, circulate and are directedtoward infection and inflammation by chemotaxis which moves cells towarda higher concentration of a given chemical. Once a neutrophil finds aforeign particle or a bacteria the neutrophil engulfs it, releasesenzymes, hydrogen peroxide and other chemicals from its granules andoxidizes it e.g. kills the bacteria. Pus at a cite of infection is deadneutrophils and other cellular debris. Although this process is healthyin fighting infection, the body generally over reacts and causesadditional inflammation due to excessive oxidation see U.S. Pat. No.5,211,937 and in particular FIG. 5 thereof.

Compounds of the invention can decrease the rate of and/or overallamount of the oxidation and thereby reduce inflammation. A CGN compoundof the invention may provide advantages over conventional nitroxides forexample 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempoll) such asthe following:

1) Target delivery: CGN ester penetrates the stratum corneum and ispreferentially taken up by viable skin cells following topicalapplication. Upon entering the cell, the ester bonds of the prodrug arecleaved by intracellular esterases, converting the CGN to its free acidform and making it much less membrane permeable. This in vivo enzymaticconversion effectively compartmentalizes the CGN inside the esterasecontaining skin cell.

2) In vivo gating function: In its hydrolyzed free acid state, thecarboxylate serving as the “gate”, will protect or safeguard theintracellular catalytic activities of nitroxide.

3) In vivo safety: Acute toxicity (LD₅₀) of CGN, unlike Tempol (LD₅₀=375mg/kg), was not detectable at 2 g/kg.

In short, the invention discloses CGN with a intramolecular carboxylatemoiety that “safeguard” the in vivo reduction of its nitroxide moietywithout affecting its catalytic activities.

The present invention discloses compounds, formulations and methods ofuse of carboxylate-gated nitroxide (CGN) for the prevention andtreatment of diseases or injuries arising from reactive oxygen spices(ROS). Compounds, formulations and methods of the invention may be usedalone or in combination with compounds disclosed in any of U.S. Pat.Nos. 5,725,839; 5,741,893; 5,767,089; 5,804,561; 5,807,831; 5,817,632;5,824,781; 5,840,701; 5,591,710; 5,789,376; 5,811,005; 6,048,967.

The present invention encompasses general classes of CGN includingmono-function CGN and bi-function CGN. Mono-function CGN refers to itsanti-ROS injury activity alone. Bi-function CGN refers to the additionof a 2^(nd) therapeutic activity from the conjugated existing drugmoiety CGN not only provide “carboxylate-gate” effect but in itsestablished form also targets delivery its intracellular anti-ROSactivity. The in vivo targeted delivery and stability of CGN enhancesits therapeutic index and diagnostic utility.

Stable mono- and bi-function CGN compounds including their esterifiedderivatives and precursors thereof alone and in formulations and theuses of such are disclosed. As mono-function CGN the oxidation/reductionactivity and compartmentalization can be regulated by the esterificationof the carboxylic acid group(s). Compounds of the invention comprise oneor more carboxylate yields a “gate” function which in a “prodrug” (e.g.ester) form provide increased permeability of CGN across cell membranesand also result in an enhanced in vivo half-life. The CGN may bemodified with existing drugs via ester or amide groups to formbi-function CGN.

Compositions for treating tissue damage from ROS injury contain CGN, itsesters or active drug derivatives thereof, in a suitable pharmaceutical,cosmetic, and diagnostic formulation for intravenous, intra-peritoneal,intra-muscular, oral, topical, nasal, pulmonary, vaginal, transdermal orrectal administration.

Compound Synthesis:

Three general structure formulae of CGN are shown in FIGS. 1, 2 and 3.Structure I is CGN described in FIG. 1. It is synthesized from thereaction of any keton or aldehyde nitroxide with esterifieddi-carboxylic acid. Structure II is mono-amino CGN described in FIG. 2.It is synthesized from the reaction of carboxyl or acetamido nitroxidewith di-esterified dicarboxylic α-amino acid. Structure III is adi-amino acid CGN (see FIG. 3). It is synthesized from carboxyl oracetamido nitroxide with esterified and amidio di-amino acid, whereinesterified group is any compound with an OH function group includingexisting drug. In structure III the two amino groups serve to link anitroxide and, optimally, other moieties with a carboxylic group. Thus,examples of CGN include esterified di-carboxylate CGN, esterifiedmono-amino acid CGN, and di-amino acid CGN and other derivatives,including di-nitroxide, tri-nitroxide derivatives, or conjugates usedalone or in combination with existing drugs or targeting molecules.

The compositions and routes of administration of CGNs are described.Among many examples topical applications of compositions of theinvention show how the targeted delivery of the CGN intracellularly isachieved through cellular esterase cleavage of hydrophobic esterifiedCGN as a pro-drug to a hydrophilic CGN in its free acid form. A secondexample of CGN such as E-TOPS has been tested for plasma half-life inco-administration as compared with a prior art nitroxide, Tempol. Aprolonged half-life of E-TOPS over Tempol was observed when E-TOPS andTempo were administrated (i.p, i.v., i.m., and oral). The third exampleof CGN such as DE-TOPS have been tested in hairless mouse model ofUVB-induced skin damage.

Definitions

The term “nitroxide” is used herein to describe molecules comprising anoxygen and a nitrogen atom directly bound to each other. A nitroxide maybe and electron donor or acceptor. Nitroxides may comprise stablenitroxyl free radicals including precursors (such as the N—H form), andderivatives thereof including their corresponding hydroxylaminederivative (N—OH) where the oxygen atoms are replaced with a hydroxylgroup and exist in a hydrogen halide form. Nitroxides of the inventionmay be administered to a system, such as a human, and act to modulateoxidation and reduction reactions by donating or accepting an electronStability of the unpaired electron of the nitroxide is provided at thenitrogen nucleus by two adjacent carbon atoms that are substituted withstrong electron donor groups. With the partial negative charge on theoxygen of the N—O bond, the two adjacent carbon atoms together localizethe unpaired electron on the nitrogen nucleus. Nitroxides generally mayhave either a heterocyclic or linear structure. A nitroxide of theinvention may have one, two or more caryboxyl groups or groups such asester groups which can be readily changed to carboxyl groups whichgroups aid in maintaining the free electron which allows the nictroxideto act as an electron donor. In an in vivo environment a nitroxide mayreact with a first superoxide to form oxoammonium (as an electron donor)and then react with a second superoxide to re-form the nitroxide (as anelectron acceptor). If a nitroxide is reduced to a hydroxylamine itloses its ability to modulate reactions. By positioning the nitroxidebetween two carboxylic acid groups a “gating” effect is obtained, i.e.the nitroxide is protected and its ability to modulate reactions ismaintained over a longer period of time in a greater range of in vivoenvironments as compared to a molecule lacking the carboxylic acidgroups.

The terms “treat,” “treatment” and the like are used herein to generallymean obtaining a desired pharmacological and/or physiological effect. Atreatment is an approach for obtaining beneficial or desired clinicalresults which include but are not limited to decreasing undesirableeffects of reactive oxygen species (ROS). The effect may be prophylacticin terms of completely or partially preventing a disease and/or symptomthereof and/or may be therapeutic in terms of a partial or complete cureof the disease and/or adverse effect attributed to the disease. Ingeneral, methods of the invention involve treating diseases generallyassociated with an inflammatory response and may be applied to a varietyof different areas and in particular membrane surfaces including theskin, mucus membranes including those in the GI tract, nose, throat,mouth, vaginal cavity, ocular surfaces, as well as the surfaces of thelungs and the surfaces of the vascular system. “Treatment” as usedherein covers any treatment of such a symptom or disease in a mammal,particularly a human, and includes:

(a) preventing or diagnosing the disease and/or symptoms in the subjectwhich may be predisposed to the disease and/or symptom but has not yetbeen diagnosed as having it;

(b) inhibiting the disease, i.e. arresting it's development; and/or

(c) relieving the disease and/or it's symptom, i.e. causing regressionof the disease and/or the symptoms caused by the disease.

The invention is directed towards modulating the inflammatory responseand in particular, modulating excessive oxidation. The inflammatoryresponse and the accompanying excessive oxidation associated with suchcan be caused by a variety of physical traumas including subjecting thecells to all types of radiation including ultraviolet and nuclearradiation as well as blunt trauma. The treatment may be combined withother co-treatments such as using anti-inflammatory drugs or antibioticdrugs to modulate the inflammation and prevent infection.

Types of treatment which might be carried out using compounds andcompositions of the invention including applying the compounds and/orcompositions to the skin in order to treat sunburn before it occursand/or treat the inflammatory response related to sunburn after thesunburn has taken place. Further, the patient's skin may be treatedafter and/or before the application of radiation used in the treatmentof cancer or fluoroscopy. In a similar manner a patient may be allowedto gargle a mouthwash containing compounds of the invention in order totreat the adverse effect of radiation on the internal surfaces of themouth and throat. Compounds of the invention may be applied directly tothe bone marrow in order to provide for protection against excessiveoxidation in the bone marrow. Compounds of the invention may be injectedinto a patient in order to modulate the inflammatory response andexcessive oxidation in the vascular system. Compounds of the inventionmay be used in treatment when being included within a cosmeticformulation applied to the skin. Further, when the skin is subjected tocosmetic surgery and/or laser resurfacing compounds and formulations ofthe invention may be applied prior to the laser resurfacing and/or afterthe laser resurfacing in order to modulate the inflammatory response.These and other types of treatment will occur to those skilled in theart upon reading this disclosure.

The term “effective amount” is an amount sufficient to effect abeneficial or desired result including a clinical result, and as such an“effective amount” depends on the context in which it is being applied.An effective amount can be administered in one or more doses. Aneffective amount may be an amount sufficient to obtain treatment e.g.modulate undesirably high levels of reactive oxygen species.

“Comprising” and its cognates mean “including.”

Molecular Characteristics

The physiological compartmentalization or site directed delivery of CGNpursuant to this invention can be achieved through several discretechemical structures or molecular modifications. The molecules aredesigned pursuant to the criteria disclosed herein to provide theselected permeability and reactivity characteristics. Modified CGNcompounds may be topically applied and targeted to specific cells e.g.viable cells of the epidermis. The membrane solubility of the nitroxideis altered by converting the CGN to the modified form as describedherein. For example, ester groups are added making the molecule morelipid soluble. These groups are removed by reactions catalyzed bycellular esterase making CGN more water-soluble and less lipid soluble(e.g. less soluble in a human lipid by 1 log, 2 logs or 3 logs or more).Once the modified CGN has entered the cell, the ordinary intracellularhydrolysis mechanisms of endogenous esterase create derivatives of CGN,which have reduced membrane permeability. Thus, these compounds readilyenter the cell (when lipid soluble), but resist leaving the cell (whenmore water soluble and less lipid soluble), and as a consequence,exhibit increased permeability for transmembrane entry into a viablecell. The decreased lipid solubility and increased water solubility alsoprovides a decreased tendency of the nitroxide to seep out of the viablecells or tissue e.g. by ordinary physiological clearance processes. Thisfeature yields selectable preferential compartmentalization in vivo anda sustained therapeutic or diagnostic potential.

As described herein, accumulation and sequestration orcompartmentalization of CGN may be enhanced by esterification. Topicalapplications prefer a diester, which may be asymmetric, and where oneesterified group is made more labile than the other. For example, thet-butyl esters of BE-TOPS will be more readily hydrolyzed than ethylester because it is more labile than ethyl ester. Hydrolysis of anasymmetric nitroxide comprising these two esters will thus first yield aless membrane permeable mono-carboxyl E-TOPS. The selection of theparticular ester di-carboxylic acid derivatives, e.g., succinate,determines the compartmentalization and intracellular accumulationcharacteristics and thus may be tailored to be higher or lower forspecific diagnostic or therapeutic indications. Likewise, preparation ofdi-esters of naturally occurring dicarboxylic amino acids (eg aspartate,glutamate)will permit increased accumulation and sequestrationintracellularly and have an added advantage in that naturally occurringamino acids are well known in administration, distribution, metabolismand excretion (ADME) studies.

The CGN that can be employed in this invention are structurally diversebecause the requisite property of the CGN is its ability to beesterified. Thus, CGNs in their monocarboxylic, dicarboxylic, orpolycarboxylic state may be employed. Selected embodiments of thepresent invention have the following structures, although the inventioncontemplates derivatives, isomers, substitutions, polymers, and otherroutine chemical modifications that preserve the functionality herein.

Compounds of the invention include those encompassed by generalstructural formula I:

FIG. 1

wherein R₁ is any nitroxide or more specifically any nitroxyl radical(NO) containing group with one unpaired electron localized around thenitrogen atom or more specifically a linear (straight or branched chain)or cyclic nitroxyl radical further comprising carbon and hydrogen atoms.An example of an R₁ is 2,2,6,6-tetramethyl-4-piperidene-1-oxyl;

wherein R₂ and R₃ are each independently hydrogen, alkyl, alkeynyl,aryl, aralkyl, akaryl or a nitroxide and in particular may independentlybe hydrogen, or ethyl, it should be noted that when R₂ and/or R₃ are H acarboxylic acid group is formed and the acid may be converted to a saltin the appropriate environment e.g. in a human body. Any salt may beformed and in particular Na and K salts may be formed; and

wherein “n” is an integer of from 0 to 18 (e.g. 1–6 or 1–4) and inparticular can be “1,” “2” or “3.”

In the structure I above the “R₁,” group may be bound via a —NH— groupto provide a structure of the invention as shown in formula II below:

FIG. 2

wherein the variables R₁, R₂, R₃ and n are as defined above and inparticular “n” may be 1, 2 or 3. Further, n may be “1” when “R₁” is asingle ring cyclic moiety comprising an “NO” group with one unpairedelectron localized around the nitrogen atom, e.g. R₁ may be2,2,6,6-tetramethyl-4-coboxyl piperidene-1-oxyl.

In the structure I or II above either the “R₂” or “R₃” may be bound notto the “O” but to an “NH” connecting moiety. When the “R₂” and “R₁” areeach connected by an “NH” the resulting structure III as shown below isobtained.

FIG. 3

Compounds of the invention are covered by the structure III above whenthe variables R₁, R₂, R₃ and “n” are as defined above for I and II.

In each of the structures I, II and III the R₁ moiety is defined and maybe a nitroxyl radical with one unpaired electron which nitroxyl radicalmay be a cyclic nitroxyl as shown in structure IV below.

Compounds of the invention are exampled by the general structuralformula IV:

FIG. 4

wherein R₂ and R₃ are each independently a moiety as defined aboverelative to any of the structures I, II, or III and in particular R₂ andR₃ may be methyl, ethyl, or butyl which may be tertiary butyl.

As used herein the circle can be any cyclic moiety with the —N— of theNO group at at least one position. The circle can be a six membered ringas in FIG. 5, a five membered ring as in FIG. 13 or other ring and/orfused ring structure.

In one structure where R₂=R₃=H (TOPS) in FIG. 7 is the structure withinin viable cell after esterase cleavage which gives the efficacy ofnitroxide intracellularly. In one embodiment the R₂ and (or) R₃ arethemselves structures as per FIG. 11 and FIG. 12 FIG. 6 describesasymmetric structure with R₂=H and R₃ is each any moiety as definedabove with respect to the structures of I, II and III. FIG. 8 gives anexample of the FIG. 6 where R₃=ethanol (E-TOPS). FIG. 9 and FIG. 10gives an example of symmetric structure of FIG. 5 where R₂=R₃=ethanol(DETOPS) or R₂=R₃=t butanol (DTTOPS). FIG. 11 gives an example ofdi-radical structure where R₂=Tempol (TETOPS).

In any structure of a compound of the invention the R₂ and R₃ may beused to bind the molecule to other atoms, molecules, or biologicalmaterial. For example, in structure III the R₂ may be used to bind themolecules to other reactive cites as described in any of U.S. Pat. Nos.5,725,839; 5,741,893; 5,767,089; 5,804,561; 5,807,831; 5,817,632;5,824,781; 5,840,701; 5,591,710; 5,789,376; 5,811,005; 6,048,967.Further, the NH connected to the OR₂ group may be used to bind to othermolecules such as a copper tri-peptide.

Since the compounds described herein provide a targeted therapeutic doseof antioxidant nitroxide, they are highly effective in preventing andalleviating the effects of oxidative stress. E-TOPS was found to have anincreased in vivo half-life compared to Tempol via intravenousadministration, intramuscular, oral, and intraperitoneal administrationat 200 mg/kg. See FIGS. 18, 19, 20, and 21. The E-TOPS has also beendemonstrated to have a very low acute toxicity profile compared toTempol in an LD₅₀ model in mice (see FIG. 24).

In transdermal applications, the DE-TOPS (FIGS. 32, 35) formulationreduces UVB light induced skin thickening, hemorrhage, inflammation whenapplying pre and post radiation in mice. DE-TOPS also shows the effecton chronic lesion induced by UVB radiation (FIG. 33). These formulationsalso compare favorably to Retin A to reduce winkling (see FIG. 37). Asdescribed in more detail below, a major advantage of the CGN compoundsof the present invention is the ability to administer a physiologicallycompatible solution in a variety of routes. Also, depending on thetarget cells and the diagnostic or therapeutic goal, the activity ofthese compounds may be enhanced by concurrent subcutaneousadministration of a polynitroxide.

Compositions of the invention may be used indermatologically/cosmetically acceptable vehicles for transdermal suchas ointments, lotions, or gels, and other solvents or carriers acting asa dilutant, dispersant or carrier. The vehicle may comprise materialscommonly employed in skin care products such as water, liquid or solidemollients, silicone oils, emulsifiers, solvents, humectants,thickeners, powders, propellants and the like.

As noted above, the unpaired nitroxyl electron gives nitroxides otheruseful properties in addition to the antioxidant activity. Inparticular, nitroxides in their free radical form are paramagneticprobes whose EPR signal can reflect metabolic information in biologicalsystems, e.g., oxygen tension or tissue redox states. Because naturallyoccurring unpaired electrons are essentially absent in vivo, EPR imaginghas the further advantage that there is essentially no background noise.Nitroxides also decrease the relaxation times of hydrogen nuclei, andare useful as contrast agents in proton or nuclear magnetic resonanceimaging (MRI). Nitroxides can also act as contrast agents to addmetabolic information to the morphological data already available fromMRI. For example, by substituting various functional groups on thenitroxide, it is possible to manipulate properties including relaxivity,solubility, biodistribution, in vivo stability and tolerance. Contrastenhancement obtained from nitroxide can improve the performance of MRIby differentiating isointense, but histologically dissimilar, tissuesand by facilitating localization and characterization of lesions, suchas blood brain barrier damage, abscesses and tumors.

Formulations

In view of the stable chemical nature of the nitroxides, thecompositions disclosed herein can be administered to a patient byvarious routes. For the purposes of this invention, “pharmaceutical” or“pharmaceutically acceptable” compositions are formulated by knowntechniques to be non-toxic and, when desired, used with carriers oradditives that are approved for administration to humans. As describedin the Examples below, the routes of administration include any type ofinjection including intramuscular, subcutaneous or intraperitoneal;intravenous, ocular or intraocular, oral; any type of transmucosaldelivery including, bucal, nasal, anal, and vaginal; topical ortransdermal; and intrapulmonary. Such compositions may include buffers,salts, or other solvents known to these skilled in the art to preservethe activity of the vaccine in solution.

A composition or formulation of the invention may comprise an effectiveamount of a nitroxide of the invention in a pharmaceutically acceptableexcipient or carrier. The carrier is generally a relatively inertsubstance that facilitates administration of a pharmaceuticallyeffective substance in a a dosage form suitable for delivery to apatient e.g. a human. Suitable excipients include by are not limited tostabilizing agents, wetting and emulsifying agents, salts for varyingosmolarity, encapsulating agents, buffers, skin penetration enhancers,skin creams, and gels. Examples of pharmaceutically acceptableexcipients are described in Remington's Pharmaceutical Sciences (AlfonsoR. Gennaro, ed., 19^(th) edition, 1995) incorporated here to discloseand describe carriers and formulations.

Topical formulations for application to the skin may comprise anitroxide, a carrier, and sun block or UV absorber. A large number of UVabsorbers are known as disclosed in any of U.S. Pat. Nos. 6,235,271;6,224,854; 6,071,501; 5,976,513; 5,972,316; and 5,968,485 and patentsand publications cited in these patents. The UV absorber may be anyknown oil-soluble organic UV absorber, especially those which arealready approved and marketed for cosmetic use. Such oil-soluble organicUV absorbers are described, for instance, in “Sunscreens”, Development,Evaluation and Regulatory Aspects, Eds.: N. J. Lowe and N. A. Shaath, M.Dekker Inc., New York and Basel, 1990; and Ken Klein, Encyclopedia of UVabsorbers for sunscreen products, Cosmetics & Toiletries 107 45–64(1992).

The oil-soluble, non-micronised UV absorber may be, for example, ap-aminobenzoic acid derivative such as an ester, salt or anamine-modified derivative of p-aminobenzoic acid; a salicylic acidderivative such as an ester or salt thereof; a benzophenone derivative;a dibenzoylmethane derivative; a diphenylacrylate derivative; abenzofuran derivative; a polymeric UV absorber containing one or moresilico-organic residues; a cinnamate ester; a camphor derivative; atrianilino-s-triazine derivative; phenylbenzimidazole sulfonic acid andits salts; urocanic acid (3-imidazol-4-yl-acrylic acid) or its ethylester; menthyl anthranilate; a benzotriazole; a hydroxyphenyltriazinederivative; or a bis-resorcinol-dialkylaminotriazine.

Specific examples of benzophenone derivatives includebenzophenone-3-(2hydroxy-4-methoxybenzophenone), benzophenone-4(2-hydroxy-4-methoxybenzophenone-5-sulfonic acid) andbenzophenone-8-(2,2′-dihydroxy-4-methoxybenzophenone).

A specific example of a dibenzoylmethane derivative is butylmethoxydibenzoylmethane[1-(4-tert.-butyl)-3-(4-methoxyphenyl)propane-1,3-dione].

Specific examples of a diphenylacrylate derivative include octocrylene(2-ethylhexyl-2-cyano-3,3′-diphenyl acrylate) and etocrylene(ethyl-2-cyano-3,3′-diphenyl acrylate).

Specific examples of a benzofuran derivative include the3-(benzofuranyl)-2-cyanoacrylates described in U.S. Pat. No. 5,338,539or EP 582189, the 2-(2-benzofuranyl)-5-tert.-butylbenzoxazoles describedin U.S. Pat. No. 5,518,713 and the 2-(p-aminophenyl)benzofuransdescribed in U.S. Pat. No. 5,362,481.

Specific examples of a polymeric UV absorber containing one or moresilico-organic residues are the benzylidenemalonate silicone derivativesdisclosed in EP 709080 in which R.sub.15 is H or OMe and r isapproximately 7; and the polymers of the benzotriazole silicone typedescribed in WO 94/06404.

Specific examples of a cinnamate ester include octyl methoxy cinnamate(4-methoxycinnamic acid 2-ethylhexyl ester), diethanolamine methoxycinnamate (diethanolamine salt of 4-methoxycinnamic acid), isoamylp-methoxycinnamate (4-methoxycinnamic acid 2-isoamyl ester),2,5-diisopropyl methyl cinnamate, the cinnamido derivatives disclosed inU.S. Pat. No. 5,601,811 and the derivatives described in WO 97/00851.

Specific examples of camphor derivatives are 4-methyl-benzylidenecamphor [3-(4′-methyl)benzylidene-bornan-2-one], 3-benzylidene camphor(3-benzylidene-bornan-2-one), polyacrylamidomethyl benzylidene camphor{N-[2(and 4)-2-oxyborn-3-yliden-methyl)benzyl]acrylamide polymer},trimonium benzylidene camphor sulfate[3-(4′-trimethylammonium)-benzylidene-bornan-2-one methyl sulfate],terephthalydene dicamphor sulfonic acid{3,3′-(1,4-phenylenedimethine)-bis-(7,7-dimethyl-2-oxo-bicyclo-[2.2.1]heptan-1-methanesulfonicacid} and salts thereof and benzylidene camphor sulfonic acid[3-(4′-sulfo)benzylidene-bornan-2-one] and salts thereof.

Specific examples of trianilino-s-triazine derivatives include octyltriazine [2,4,6-trianilino-(p-carbo-2′-ethyl-1′-oxy)-1,3,5-triazine, thetrianilino-s-triazine derivatives disclosed in U.S. Pat. No. 5,332,568,the trianilino-s-triazine derivatives described in EP 517104,trianilino-s-triazine derivatives disclosed in EP 570838, thetrianilino-s-triazine derivatives described in U.S. Pat. No. 5,252,323,the trianilino-s-triazine derivatives described in WO 93/17002-A1 andthe trianilino-s-triazine derivatives disclosed in WO 97/03642-A1.

A specific example of a benzotriazole is2-(2-hydroxy-5-methyl-phenyl)benzotriazole.

Specific examples of hydroxyphenyltriazine derivatives include, e.g.those described EP-A1-775,698, such as2,4-bis-{[4--(2-ethyl-hexyloxy)-2-hydroxy]-phenyl}-6-(4-methoxyphenyl)-1,3,5-triazine.

Specific examples of bis-resorcinol-dialkylaminotriazines are, e.g.,those described in EP-A1-780,382.

The inorganic micropigment UV absorber, the optional component b) of thenew sun protection agent may be, for example, titanium dioxide coatedwith aluminium oxide or silicon dioxide, zinc oxide coated withaluminium oxide or silicon dioxide, or mica.

The polymeric hollow sphere component, component c) of the new sunprotection agent according to the present invention, may be, forinstance, those described in EP-A-761,201.

Methods

A method of treatment is disclosed whereby a nitroxide compound isadministered to a patient in a therapeutically effective amount. Thenitroxide compound is comprised of a first portion which comprises anitrogen atom and an oxygen atom bound directly together and an unpairedelectron (•NO), and a second portion which provides a negative charge.The first and second portions are connected directly or indirectly by alinking group positioned between and bound to the first portion and thesecond portion in a manner such that the negative charge of the secondportion stabilizes the •NO of the first portion when the nitroxide ispresent in the patient. By stabilizing the •NO portion of the moleculethe nitroxide is allowed to interact with reactive oxygen species in thepatient for a longer period of time and modulate adverse effects ofthose reactive oxygen species.

A method is disclosed whereby a formulation is applied to the skin of apatient which may be a human. The formulation is comprised of a carrierand a compound which has a first lipid solubility and a first aqueoussolubility. The compound is allowed to permeate the patient's skin andreach viable cells and react in the presence of enzymes of the cells.The reaction results in a compound which has a second lipid solubilitywhich is substantially less than the first lipid solubility and alsoobtain a second aqueous solubility which is substantially greater thanthe first aqueous solubility thereby allowing the compound to permeateto viable cells and remain in contact with those cells. The compound maybe a nitroxide which aids in modulating adverse effects of reactiveoxygen species.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1 Synthesis and Preparation of Mono and Dicarboxylic Acid andEsterified Nitroxide Species

The following chemical synthesis protocols yield stable nitroxide freeradicals whose physiological compartmentalization, as a function ofmembrane permeability and clearance in vivo, is regulated by anegatively charged anion such as mono- or di-carboxylic acids. Topicalapplications are particularly advantageous with ester derivatives thatprovide differential permeability across hydrophobic barriers with afirst nitroxide species having increased membrane permeability relativeto a second species having increased intracellular retention andantioxidant therapeutic utility after hydrolysis by intracellularesterases.

(a) Synthesis of Monoethyl andDiethyl2,2,6,6,-Teteramethyl-1-oxyl-4-piperidinyl succinate (E-TOPS) and(DE-TOPS).

A dry, two-necked flask fitted with a reflux condenser and magneticstirrer is charged with 45 ml of absolute tert-butanol and 6.72 g ofpotassium tert-butoxide under nitrogen atmosphere. The mixture is boiledand heated under reflux until all solids are dissolved. The flask isthen cooled and 6.8 grams of 4-oxo-[TPO], 12 ml of diethyl succinate and15 ml of tertiary butanol is added. The reaction mixture is then heatedfor 10 minutes. After cooling with ice, and neutralizing with diluteHCL, the bulk of the alcohol is distilled off under reduced pressure.The residue is poured into 350 ml of ice water and acidified with dilutehydrochloric acid to pH 3, and extracted with methylene chloride. Thecombined extracts are washed several times with a 1% ammonia solution.The solutions are cooled with ice, acidified and extracted again withmethyl chloride. The resulting extract is then dried with sodiumsulfate. After evaporation of the methylene chloride, the remaining oilyred liquid is triturated with hexane. The crystal of the monoester ispressed on a porous porcelain plate and recrystallized from a mixture ofether and hexane. The product is a yellow prism with a melting point of103° C., and the expected yield is in the 60–70% range.

FIG. 14 shows the separation of TOPS, E-TOPS, and DE-TOPS by liquidchromatography.

Example 2 Hydrolysis of DE-TOPS to TOPS

Referring to FIG. 15, the hydrolysis of DE-TOPS to the non-esterifiedTOPS form will yield selective cell membrane permeability and increasedintracellular retention when the nitroxide compounds are exposed toesterases or any intracellular enzyme or other biochemical reaction thatcleaves the ester group. The application of DE-TOPS as a hydrophobicpro-drug will penetrate the stratum corneum (dead cells) into themetabolically active base-membrane layers. Enzymatic hydrolysis ofDE-TOPS will allow the product TOPS to be retained in the aqueous phaseand hopefully and primarily in the intracellular volume. To demonstratethis reaction, chemical hydrolysis of DE-TOPS is shown to yield acompound which preferentially distributes in water vs. octanol.

A 20 mg sample of DE-TOPS was added to a mixture of 1 ml water and 1 mloctanol and allowed to partition. After 15 minutes, a 40 μl sample ofwater or oil was taken for EPR spectral analysis. Next, 20 mg of DE-TOPSwas mixed in 1 ml of 10 mM NaOH and allowed to incubate overnight atroom temperature. The solution was neutralized with hydrochloric acidand added to 1 ml of water and 2 ml of octanol. The mixture was allowedto partition, and after 15 minutes, a 40 μl sample of water or oil wastaken for EPR spectral analysis. EPR spectral were taken using a VarianE9 spectrophotometer. Sweep width was 100 G, frequency was 9.535 GHz,microwave power was 2 mV and the modulation frequency was 100 Hz.

Before hydrolysis, DE-TOPS was preferentially distributed in octanol.The partition coefficient (LogP) for DE-TOPS was found to be 1.7. Afterhydrolysis, the presumed product TOPS partitions in the aqueous phase.The partition coefficient (LogP) for TOPS was found to be −0.9. Thisshows that following hydrolysis, the product thus formed issignificantly hydrophilic and would be more readily compartmentalizedintracellularly.

Example 3 Synthesis of TE-TOPS

This pilot study is to synthesize TE-TOPS as the proof of Tempol,excising compound may be incorporated into the TOPS-ester prodrugconstruct. TE-TOPS was synthesized from the ¹⁴N-E-TOPS and ¹⁵N-Tempol.The synthesis is a multi-step process involving condensation ofoxo-Tempo (4-oxo-2,2,6,6-tetramethyl-piperidine-1-oxyl) withdiethyl-succinate followed by esterification of the ¹⁴N-E-TOPS witheither ¹⁵N-Tempol or ethanol, yielding ¹⁴N-DE-TOPS or ¹⁴N-,¹⁵N-TE-TOPS,respectively. The reaction to form the bi-radical ¹⁴N, ¹⁵N-TE-TOPS wasmonitored by both HPLC and EPR. ¹⁴N-E-TOPS gives a triplet EPR spectrumas shown in FIG. 16A left panel. Its HPLC elution time is 6.3 min asshown in FIG. 16, right panel. ¹⁵N-Tempol gives a doublet EPR spectrumas shown in FIG. 16B, left panel. The HPLC elution time for ¹⁵N-Tempolis 5.3 min as shown in FIG. 16B, right panel. After 3 hours reaction,the height of the HPLC peaks at 5.3 and 6.3 min decreased while a newpeak at 13.9 min appeared (see FIG. 16C, right panel). After 6 hours ofreaction, ¹⁵N-Tempol and ¹⁴N-E-TOPS peaks almost disappeared (FIG. 16D,right panel). To ensure the HPLC peak at 13.9 min consists of ¹⁴N-,¹⁵N-TE-TOPS, the peak was collected then assayed by EPR. The EPRspectrum is shown in FIG. 16D, left panel. The broadening and appearanceof new peaks are due to spin-spin and spin-spin dipole interactionsbetween the bi-radicals.

Example 4 Hydrolysis of TE-TOPS to TOPS and Tempol

20 mg of ¹⁴N-¹⁵N-TE-TOPS was mixed in 1 ml of 10 mM KOH and allowed toincubate overnight at room temperature. The solution was neutralizedwith hydrochloric acid. EPR spectral were taken using a Varian E9spectrophotometer. Sweep width was 100 G, frequency was 9.535 GHz,microwave power was 2 mV and the modulation frequency was 100 Hz. Asharper EPR spectrum in FIG. 17A indicated that TE-TOPS was fullyhydrolyzed in comparison with the broad spectrum in FIG. 16D left panel.¹⁵N-¹⁴N-TE-TOPS in topical cream were applied on mouse skin. The urinesample showed the esterase cleavage in vivo see FIG. 17B

Example 5 In Vivo Plasma Half-Life of DE-TOPS and Tempol

As noted above, a principal drawback in existing nitroxide-basedcompositions for in vivo therapeutic or diagnostic use is the limitedhalf-life of these molecules and their rapid in vivo bioreduction andclearance. The result of the comparatively short half life of Tempol isa need to administer larger and larger doses to yield a profoundtherapeutic or diagnostic effect. As shown in FIG. 24 below, theincreased dosages of nitroxides can yield acute and chronic toxicity.However, where the plasma half-life of a compound is increased, theoverall dosage in both acute and chronic indications can be reduced.

Plasma half-life is measured by collecting the spectrum each minute for60 to 90 minutes. The peak height of ¹⁵N Tempol or ¹⁴N E-TOPS EPR signalis calculated from each spectrum. The peak height of TempolL or E-TOPSis plotted against time as shown in FIGS. 18–21 for i.p., i.v., i.m.,and oral respectively. FIG. 18 shows a measurement of the in vivo plasmahalf-life of an i.p.administration of E-TOPS and Tempol at 125 mg/kg.¹⁴N E-TOPS (upper line) is substantially enhanced for the entirehalf-life of the compounds. Although ¹⁵N Tempol (lower line) has ameasurable half-life exceeding 40 minutes, E-TOPS has substantiallyhigher activity for at least 80 minutes. The doses are 125 mg/kg ofE-TOPS and 80 mg/kg Tempol at a 1:1 molar ratio.

FIG. 19 shows a measurement of the in vivo plasma half-life of anintravenous administration of E-TOPS and Tempol at 125 mg/kg. In theintravenous infusion example, Tempol (lower line) is rapidly reduced invivo such that, by the five-minute mark after infusion, very littleactive Tempol remains in the intravascular space.

As is shown in FIG. 20, beyond the first few minutes the in vivo plasmahalf-life of E-TOPS is dramatically extended over Tempol for at least 60minutes following intramuscular co-administration.

Referring to FIG. 21, the in vivo plasma half-life of E-TOPS compared toTempol is shown to be extended when both compounds are administeredorally. As in FIGS. 18–21, the doses are 125 mg/kg of E-TOPS and 80mg/kg of Tempol at 1:1 molar ratio

Example 6 Penetration and Compartmentalization in Human Skin

Referring to FIG. 22, DE-TOPS (100 mM) in a petroleum base was appliedon fresh human skin. The receptor buffer is PBS with 0.01% sodium azide.The buffer was constantly stirring. The cell was maintained at 37 C.with a circulating water bath. FIG. 22 shows the degree of skinpenetration. Mouse skin (or donor human skin) to cover the top of thereceptor and cell DE-TOPS is applied on top of the skin. Under the skin,PBS buffer is applied to keep the skin alive. 24 hours later the bufferwill be collected for EPR assay. The surface of the skin is cleaned andthe skin sample tested for EPR Signal. Although Tempol and DE-TOPS havesame signal intensity prior to application, twenty-four hours afterapplication a stronger Tempol signal exists in the buffer compound toDE-TOPS. However, in the skin the E-TOPS signal is stronger than Tempol.Thus, DE-TOPS is localized in skin compared with Tempol. DE-TOPS willpenetrate into the cells of the epidermis and dermis where it will beenzymatically hydrolyzed and become compartmentalized. Compared to thefreely soluble Tempone, this would result in a more uniform distributionof DETOPS in these skin layers.

Preliminary S-band EPR spectroscopy and imaging experiments using DETOPSwere performed on the skin of a human volunteer. The human volunteer'sforearm skin, about 6 mm diameter circular spot, typically at the lunarsurface of the wrist, was washed thoroughly with alcohol and 3 μL of 100mM DE-TOPS solution (about 2×10¹⁷ spins) was applied to the marked skinarea. Five minutes later, when the deposited solution dried, a speciallydesigned positioning holder with a 7 mm diameter well and bottom diskthat locked into a well in the resonator cap was attached to the skin tofix this region of skin to the surface resonator See, “In vivo EPRImaging of a Distribution and Metabolism of Nitroxide Radicals in HumanSkin,” He et al., J. of Magnetic Resonance 148, 155–164 (2001). EPR andEPRI measurements were then started. Measurements on the volunteer wereperformed for 15 to 20 minutes periods after which there were 30-minuterest periods in which the volunteer removed the arm from the resonatorand the magnet. This holder fixed the skin positioning and assured aconstant filling factor of the loaded resonator. The positioning holderwas left attached to the arm for the entire series of measurementslasting up to 8 hours.

Referring to FIG. 23, a color-coded image of CNO penetration andcompartmentalization in human skin is shown. The 1-D spatial images wereobtained from the skin of the fore-arms of the same human volunteer at 1hr and 8 hr post-topical application of 3 μL or DE-TOPS (100 mM inDMSO). The measurements were performed using S-band (2.2 GHz) EPRimaging system with a specially designed surface resonator as describedin He et al., J. of Magnetic Resonance 148, 155–164 (2001). The dottedline marks the surface of the skin. The estimated skin depths are markedas epidermis, dermis and subcutaneous layers. Compartmentalizationresults in a more diffuse distribution of DE-TOPS throughout the skinlayers. The 1-D EPR spatial image of DE-TOPS compared to tempone show anenhanced visual distribution throughout the dermis and epidermis byeight hours.

Example 7 Acute Toxicity LD₅₀ of Tempol and E-TOPS as Well asMetabolisms of E-TOPS

As noted above, the practical, clinical application of unbound, smallmolecule nitroxides has been limited by the reduced activity in vivo andcomparatively short in vivo half-life. The result of the reduced in vivohalf-life is the need to administer a larger dose to achieve the sametherapeutic or diagnostic effect. The toxicity of Tempol may be measuredwith an LD₅₀ model to determine the survival rate of mice at varyingdosages. FIG. 24 shows the acute toxicity curve for Tempol as a functionof survival rate with increasing dosages. The E-TOPS formulation showsessentially no decrease in survival rate at dosages up to 3.5 mmol/kgwhereas Tempol shows zero survival rate at the lower dosage of 2.0mmol/kg. Significant differences in survival rate appear between thedosage of 2.0–2.5 mmol/kg. FIG. 25 shows the stronger EPR signal(proportional to concentration) from DE-TOPS in urine compound forplasma after 6 hours, showing excretion and clearance through normalmetabolism.

Example 8 Inhibition of Superoxide Dismutases Activity of E-TOPS

Superoxide dismutase activity of E-TOPS in comparison with Tempol areshown in FIG. 26. SOD-mimetic activity of E-TOPS and Tempol was measuredby electron paramagnetic resonance. High-filed EPR peak was monitoredwith time. E-TOPS and Tempol without NADH (−NADH) curve showed nitroxidereacts with superoxide catalytically. E-TOPS and Tempol with NADH(+NADH) curves showed two-electron reduction from oxoammonium tohydroxylamine. The result showed that E-TOPS had the similar SODactivity as Tempol.

Example 9 Hemoglobin Toxicity in Cultured Rat Cortical Neurons

E-TOPS is neuroprotective in a model of hemorrhagic transformation instroke. Primary neuronal cultures were made from forebrains of fetal ratpups (embryonic day 15). The cells were dispersed by repeated mechanicaltrituration in neuronal culture medium (MEM Eagle (Sigma, M4526),supplemented with glutamine (2 mM), penicilin-streptomycine (50Units/ml-0.05 mg/ml), heat-inactivated horse serum (10%), fetal bovineserum (10%), glucose (0.5% or 28 mM). Following centrifugation (900 g; 5min), the cells were placed onto poly-L-lysine-coated 96 well plates ata density of 5×10⁶ cells/well. Hemoglobin in saline was added at 10 uMfinal concentration and ETOPS were added at 10 uM, 1 uM, and 0.1 uMfinal concentration. 24 hours after incubation neuronal viability wasquantitatively determined using the colorimetric MTT assay. MTT wasadded to each well such that the final concentration of the dye was 0.15mg/ml. Plates were then returned to the incubator for 1 hour at whichtime unincorporated MTT was removed, and the plates allowed to air dry.The purple formazan product present in viable cells was then dissolvedby adding acidified isopropanol (with 0.1 N HCl in) and the absorbanceintensity (540 nm) was measured using a 96 well plate reader. %Control=(Test A₅₄₀/Mean Control)×100%. FIG. 27 shows increased neuronalviability with the E-TOPS samples.

Example 10 TOPS or TOPS-ester Neuroprotection of Cortical NeuronsExposed to the Peroxynitrite Generator SIN-1

SIN-1 toxicity is studied in cultured rat cortical neurons based on thegeneration of peroxynitrite. The demonstration that TOPS or TOPS-esterare neuroprotective in this model, translates into nitroxide-dependentblockade of EGFR activation caused by SIN-1.

Primary neuronal cultures were made from forebrains of fetal rat pups(embryonic day 15). The cells were dispersed by repeated mechanicaltrituration in neuronal culture medium (MEM Eagle (Sigma, M4526),supplemented with glutamine (2 mM), penicilin-streptomycine (50Units/ml-0.05 mg/ml), heat-inactivated horse serum (10%), fetal bovineserum (10%), glucose (0.5% or 28 mM). Following centrifugation (900 g; 5min), the cells were placed onto poly-L-lysine-coated 96 well plates ata density of 5×10⁶ cells/well. Cytotoxicity was induced in the cellsaccording to a published procedure (Carroll et al 2000). SIN-1(3-morpholinosydnonimine, Sigma), a PN generator, was dissolved in 50 mMphosphate (pH 5.0) just prior to use, and added to each well to give thefinal concentration of 1 mM. TOPS, E-TOPS or DE-TOPS were added at theindicated concentration to each culture 15 minutes prior to SIN-1.Neuronal viability was quantitatively determined using the colorimetricMTT assay. MTT was added to each well such that the final concentrationof the dye was 0.15 mg/ml. Plates were then returned to the incubatorfor 1 hour at which time unincorporated MTT was removed, and the platesallowed to air dry. The purple formazan product present in viable cellswas then dissolved by adding acidified isopropanol (with 0.1 N HCl in)and the absorbance intensity (540 nm) was measured using a 96 well platereader. % Control=(Test A₅₄₀/Mean Control)×100%.

Referring to FIG. 28, TOPS or TOPS-esters prevented the toxicity ofSIN-1 in a dose dependent manner. Neuroprotective concentrations ofthese compounds will prevent peroxynitrite-dependent EGFR activation bypreventing the covalent dimerization of receptors and their subsequentautophosphorylation.

Example 11 Antioxidant Activity by Inhibition of Nitration

To demonstrate antioxidant activity of DE-TOPS, E-TOPS and TOPS invitro, these compounds are compared with Tempol as the gold standard.Tempol was shown to prevent the nitration of 4-hydroxyphenylacetic acid(HPA) by peroxynitrite in vitro. In this preliminary experiment, %inhibition by Tempol, DE-TOPS, E-TOPS or TOPS of peroxynitrite-dependentnitration of HPA was measured.

Peroxynitrite was made by a procedure described previously. Solutions of1 mM 4-hydroxyphenylacetic acid (HPA, Sigma) were made in 100 mM sodiumphosphate at pH 6.5. Certain amount of TOPS, E-TOPS and DE-TOPS wereadded to 1 ml of HPA solution mentioned above to give a finalconcentration of nitroxide at 0.98, 3.91, 15.5, 62.5 and 250M.Peroxynitrite was added at a final concentration of 1 mM to start thenitration. Reactions were also carried out using inactive peroxynitriteas blank and zero nitroxide as positive control. The nitration wasfollowed spectrophotometrically at 405 nm. The concentration of4-hydroxy-3-nitrophenylacetate was determined spectrophotometrically(₄₃₀=4400 M⁻¹ cm⁻¹) after the pH of reaction mixtures were creased to10–11 with NaOH.

Referring to FIG. 29, 40% to 60% of HPA nitration by 1 mM peroxynitriteare inhibited by the nitroxides of the invention at 3 μM. The mechanismof inhibition by nitroxides is catalytic. As peroxynitrite is suggestedto be an important player in radiation-induced cellular damage,TOPS-esters has utility as a therapy for skin exposed to reactive oxygenspecies such as peroxynitrite.

Example 12 Cell Apoptosis Measured by Exposure to TNF-α

The UV light induced apoptosis may be measured by a model in whichapoptosis is induced by exposure to tumor necrosis factor alpha (TNF-α).As shown in FIG. 30, measurement of apoptosis severity over time forcells exposed to TNF-α plus E-TOPS is measured against a control.Cultured human Y-79 cells were maintained at pH 7.4 in culture flasks ina mixture of amphotericin/penicillin/streptomycin treated (1% v/v) RPMI1640 media with L-glutamine and 10% fetal bovine serum in an incubatorunder 10%-CO₂/90%-air brood conditions at 37 degrees Celsius and 20%humidity. E-TOPS was prepared as a 10 mg/ml formulation. From this stocksolution, 10 μl was added to 1 ml of the cell suspension solution sothat the final concentration of ETOPS was 100 μg/ml of ETOPS. HumanTNF-α (10 μg/1 ml) was used to prepare serial media dilutions to obtainTNF-α concentrations of 5.0 ng/ml. Cell densities and viability weredetermined by trypan blue exclusion assay on a Zeiss inverted microscopeto ensure cell concentrations prior to cytometric assaying. After gentlemixing of the TNF-α, vial sets at the given concentrations for 6, 24,and 48 hours, the cytokine-treated cells were resuspended in PBS. Thecells were resuspended in annexin V-FITC conjugate solution andincubated at room temperature in the dark for fifteen minutes. Bindingbuffer was then added to each of the samples to bring the cell densitiesup to approximately 1.0×10⁶ cell/ml. The binding buffer was prepared bymixing the following together: 10 ml of 1M HEPES/NaOH, ph 7.4, 30 ml of5M NaCl, 5 ml of 1M KCl, 1 ml of 1M MgCl₂, 1.8 ml of 1M CaCl₂ and 52.2ml of DDW.

Flow cytometric methods were employed to take advantage of annexin V'sreversible and calcium-dependent binding to negatively chargedphosphatidylserine (PS) residues in a 1:50 annexin to PS ratio. Assay at488 nm on a Becton-Dickinson FACStar flow cytometer was then conductedon the cells from the culture vials to determine the relativeproportions of cells that were nonviable. A two dimensional x-y contourplot was used to show populations of early apoptotic events separatefrom late apoptotic events. Compensation was set before the cytometrictrials by using an annexin-only stained population, a propidium-onlystained population, and an unstained population to delimit the ceilingsof detection in the respective FL-1/F1-2 quadrants. Total samplingsaveraged 5000 cell events counted out of sample populations averagingwell over 1.0×10⁶ cells/ml with statistics and regression analysis givenfor each set of sample quadrants using CellQuest software. Fluoresceinstained populations alone demarcated apoptotic detection while dualcounterstaining with propidium iodide indicated necrotic populations.

Example 13 Protection Against Ultraviolet Induced Skin Damage WhenApplying DE-TOPS Pre Radiation

Referring to FIG. 32, 11 days exposure to UVB causes significant skinthickening compared to normal mouse skin without UVB exposure.Histopathological skin sections reveal that topical application ofDE-TOPS dramatically reduces skin thickening induced by UVB. Topicalapplication was daily 10 minutes prior to UVB radiation. UVB dose was200 mJ/cm2 during 15 minutes. UVB lamp has spectrum distribution of290–310 nm at 80% below 310 nm at 20%. The DE-TOPS formulations are 100mM in a petroleum base. Referring to FIG. 31, with a similar protocol,skin burn is inhibited by DE-TOPS acutely. Referring to FIG. 33, samemice were kept for 4 month, skin lesion was inhibited by DE-TOPSchronically. In this example DE-TOPS was applied 2 hour before UVBradiation.

Example 14 Protection Against Ultraviolet Induced Skin Damage WhenApplying DE-TOPS Post Radiation

Same protocol was used as in example 13 except DE-TOPS applied 15 minpost radiation. The protective effect of DE-TOPS was shown in FIG. 34and the histopathology change was shown in FIG. 35.

Example 15 Protection Against Ultraviolet Induced Skin Damage inComparison with Tempol

Same protocol as in example 13 was used for this study. The effect ofDE-TOPS in protection of UVB induced skin damage was tested incomparison with Tempol when apply test article 10 min or 2 hour preradiation. The results in FIG. 36 showed DE-TOPS provide longertherapeutic window that Tempol (see FIGS. 36B and C)

Example 16 Anti-wrinkle Effect of BE-TOPS in Rhino Mouse

To determine the advantage of CGN compared to commercially availabletopical therapies, BE-TOPS, Retin-A, and a placebo were administered tothe skin of mice exposed to alternate days of UVB radiation. Treatmentwith Retin-A resulted in a reduction in skin wrinkling compared withcontrol animals (See FIG. 37). Although the photo-sensitivity of Retin-Acaused discoloration and flaking of the dorsal skin, cutaneoushistological sections of the dorsal skin also revealed Retin-A treatmentresulted in a reduction in the density of open and deep cysts.

BE-TOPS also has the advantage over Retin-A that DE-TOPS treated skin isnot UVB sensitive.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. A composition, comprising: a pharmaceutically acceptable carrier; anda compound of structural formula:


2. The composition of claim 1 further comprising: polynitroxyl albumin.3. The composition of claim 1 further comprising: polynitroxyl starch.4. The composition of claim 1, further comprising: a non-steroidalanti-inflammatory drug.
 5. The composition of claim 1, furthercomprising: an antibiotic.
 6. The composition of claim 1, furthercomprising: a copper tri-peptide.
 7. The composition of claim 1, whereinthe carrier is an injectable carrier.
 8. The composition of claim 1,wherein the carrier is an topical carrier.